Our long term goal is to develop effective immunotherapy strategies for brain tumors. Our preclinical translational cancer immunologic studies and early phase vaccination trials in the current funding period demonstrated the safety and immunological activity of our efforts to up-regulate type-1 antitumor T cell responses. We now propose to extend this work by exploring the ability of a specific set of microRNAs (miRs) to enhance the immunotherapeutic potential of these T cells. Our miR microarray analyses revealed that the miR-17-92 cluster of microRNAs (miR-17-92), which is known to promote cell survival and proliferation, is one of the most significantly over-expressed miR in murine type-1 T cells compared with type-2 cells. Our data suggest that the type-2-skewing tumor microenvironment can induce downregulation of miR- 17-92 expression in T cells, thereby diminishing the persistence and efficacy of tumor-specific T cells. Furthermore, T cells derived from miR-17-92 transgenic (TG) mice demonstrated superior type-1 phenotype with increased IFN-3 production and VLA-4 expression when compared with counterparts derived from wild type mice. Major barriers to cancer immunotherapy include the poor immunogenicity of tumor-antigens (TAs) and the short duration of T cell survival in tumor-bearing hosts. However, our data and those from others strongly suggest that miR-17-92 expression in T cells will lower the threshold for activation and enhance T cell proliferation, survival and type-1 functions. We therefore hypothesize that engineering TA-specific cytotoxic T lymphocytes (CTLs) to express the miR-17-92 cluster will provide potent and durable antitumor activity by potentiating TA-specific type-1 CTL functions. We will evaluate our hypothesis using T cells derived from mice TG for miR-17-92 and T cell receptor (TCR)-specific for hgp100 25-33 (Pmel) (miR-17- 92/Pmel-TG T cells). Specifically, we will determine whether: Aim 1: miR-17-92 overexpression enhances activation and survival of type-1 anti-tumor T cells in vitro We will test our hypothesis that transgene-mediated expression of miR-17-92 in TA-specific CTLs will increase sensitivity to TAs, survival, and type-1 function without inducing oncogenic transformation. Aim 2: TA-specific CTLs overexpressing miR-17-92 confer durable and robust type-1 immunity in vivo We will address whether miR-17-92 expression improves: 1) expansion and persistence of type-1 TA-specific CTLs;2) development of memory T cells;and 3) recall responses for CTL activity in non-tumor bearing mice. Aim 3: Adoptive transfer of T cells engineered to express miR-17-92 increases anti-tumor effects. We will determine whether ACT of miR-17-92/Pmel-TG CTLs will provide durable and effective anti-glioma immune responses compared with control Pmel-TG CTLs in mice bearing GL261 or de novo murine gliomas. These proposed preclinical studies will have significant impact on not only glioma-immunotherapy, but also the entire field of cancer immunotherapy, particularly through genetic engineering of TA-specific T cells. PUBLIC HEALTH RELEVANCE: Our long term goal is to develop effective immunotherapy strategies for brain tumors. We have translated our past preclinical findings into early clinical trials that demonstrated safety and immunological activities. The currently proposed preclinical study will evaluate an entirely novel immunotherapy approach with engineering of microRNAs in anti-tumor T cells, which we hope to translate into clinical trials in near future.